The document discusses different chromatography techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography (GC). It defines chromatography and describes the basic components and principles including the mobile phase, stationary phase, and separation of analytes. Specific details are provided on the procedure, applications, and instrumentation of TLC, HPLC, and GC. These techniques can be used for qualitative and quantitative analysis, identification and separation of unknown compounds, and purification of mixtures.

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I. Introduction
II. Classification of chromatographic methods
III. Principle of chromatography
IV. High performance liquid chromatography (HPLC)
V. Gas chromatography (GC)
VI. Thin layer chromatography (TLC)

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Definition:
Chromatography is defined as a procedure
by which solutes are separated by
dynamic differential migration process in a
system consisting of two or more phases,
one of which moves continuously in a given
direction and in which the individual
substances exhibit different mobilities by
reason of differences in adsorption,
partition, solubility, vapor pressure,
molecular size, or ionic charge density.

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Mobile Phase:
The Phase that travels through the
column (gas or liquid) – transport
sample through the column.
Stationary Phase:
Immiscible solid or liquid phase that
fixed in place in the column or on a
solid support – retain analytes within
the column.
Band or Zone:
-Area across which analyte is
distributed on column
-Zones of different analytes gradually
separate as bands progress down
column

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Sample
Mobile phase (eluant)
Stationary Phase
Detection method
Chromatogram
Thin Layer
chromatography
Column Chromatography

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 Method to separate components in a mixture based on
different Distribution coefficients between the two phases.
 Chromatography categorized on the basis of interaction
between solute and stationary phase
 Mobile phase either gas or liquid
 Stationary phase either liquid or solid
– Liq/Liq (Partition)
– Liq/Sol (Adsorption)
– Gas/Liq (Partition)
– Gas/Sol (Adsorption)
Gas
Chromatography
Liquid
Chromatography

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According to methodology
Planer
chromatography
Column
chromatography
Thin Layer
TLC
Paper
PC
HPLC GC
Electrophoresis

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Sample
Mobile
time
Response
A
B
Figure:
Schematic diagram showing the separation of
compounds A and B. and the output of the detector
response at various stages of elution
The process of:
Addition of sample
Mobile elution process
Separation mechanism
Retention time ?
Detection by, UV lamp, UV detector,
other detectors.
Eluted bands / collection
 Chromatogram? (function of retention
time versus detector response)
 Partition coefficient K’
 k’ = Cs/CM

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Principles of (TLC) TLC
Chromatography carried out on
active particulate material (silica
gel or alumina) dispersed on an
Inert support (flat glass plates)

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Basic Steps of TLC Technique
Preparation of the Plate
Sample Application
Chromatogram Development
Locating of the Spots

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 Slurry of the active material is uniformly
spread over the plate by means of a
commercially available spreader.
 Air-drying overnight, or oven-drying at
80-90 C for about 30 minutes.
 Ready to use thin layers (pre-coated plates)
are commercially available.
Preparation of the Plate

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Sample Application
1-2 cm 1-2 cm
2-2.5 cm
 Base line

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Locating of the Spots

Base line
Solvent front
Rf = b/a a
b
For Colored Compounds:

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Base line



Solvent front
a
Where is the spots
??
We do not know.
• Iodine or sulphuric acid is used for most organic mixtures.
• Ninhydrin is used for amino acids.
• 2,4-Dinitrophenylhydrazine is used for aldehydes and ketones
b
Rf = b/a
For Colorless Compounds:

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  
Applications of TLC Technique
Identification of Unknown Compounds
  

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Analysis of Reaction Mixture
  

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Chromatogram Development

 Avoid direct contact between the sample and the solvent system.
 The tank or chamber is preferably lined with filter paper.
 As the developing solvent travels up the plate, it dissolves the
sample and carries it up; the sample distributing itself between the
moving solvent and the stationary phase.

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Determination of the Purity of a Product Compound

Impurities
Product compound

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Quantitative Determination of
an Unknown Concentration
Unknown
  
 
 
Standard
conc.
Concentration
Signal
Calibration curve

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Instrumentation of HPLC
Mobile phase
reservoir
Solvent
mixing
valve
Pump
HPLC
Chart
Sample
injection
valve
Recorder
Waste
Detector

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Type Response Sensitivity
(ng/mL)
Refractive index Universal 1000
Conductimetric Selective 100
UV/visible absorption Selective 10
Mass-spectrometry Selective 0.1
Fluorescence Selective 0.001
HPLC Detector
Characteristics of Typical HPLC Detectors :

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HPLC Recorder
Mobile phase
reservoir
Solvent
mixing
valve Pump
Chart
injection
valve
Recorder
Detector
Waste

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What is the Applications of HPLC ?
Qualitative Analysis
Quantitative Analysis
Purification of Compounds
Identification of Compounds
Separation of Mixture Components
Peaks correspond to
individual components
Compound
Impurity
Authentic
Unknown

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Quantitative Analysis
0 10
5 g/mL
0 10
10 g/mL
0 10
25 g/mL
0 10
50 g/mL
0 10
75 g/mL
0 10
100 g/mL
0 10
Unknown
Concentration
Peak
hight
Calibration curve
External Standard Method

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G
C

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Instrumentation of GC
Flow meter
Gas
supply
Pressure
regulator
Flow
controller
Septum
Vent
Detector
Oven
Column
Injector
GC
Chart
Recorder


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GC Column
 Packed column
* ~ 3-6mm inner diameter tubing, 1-5
m long
* used for preparative separations or to
separate gases that are poorly
retained
* lower resolution
* small, uniform particle size decreases
Eddy diffusion (requiring higher
pressures)
 open tubular (more common):
* 0.1-0.5 mm inner dia., 10-100 m long
* 0.1-5 m thick sp coated on inner walls
* higher resolution, shorter analysis times,
greater sensitivity compared to packed columns

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 Flame Ionization Detector (FID):
column effluent
H2
air
cathode (collects
CHO+ ions)
anode
Detectors

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Detectors
 Flame Ionization Detector (FID):
– organic solutes are burned in flame producing CH
radicals and eventually CHO+
– CH
. + O
. CHO+ + e-
– CHO+ ions are collected by cathode, produces
current as the response

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Applications of GC ?
Qualitative Analysis
Quantitative Analysis
Identification of Compounds:
Peaks correspond to
individual components
Separation of Mixture Components:
Authentic
Unknown
Retention time comparsion
Pyrolysis gas chromatography
It is used for the identification of non-volatile materials (plastics,
natural and synthetic polymers, and some microbiological materials.
It is based on the fingerprint chromatogram for the sample, which
results from its thermal dissociation and fragmentation.

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Quantitative Analysis
0 10
5 ng/mL
0 10
10 ng/mL
0 10
25 ng/mL
0 10
50 ng/mL
0 10
75 ng/mL
0 10
100 ng/mL
0 10
Unknown
Concentration
Peak
hight
Calibration curve
External Standard Method

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Food Analysis
Analysis of foods is concerned with confirm the presence
and determination the quantities of the analytes (lipids,
proteins, carbohydrates, preservatives, flavours, colorants,
and also vitamins, steroids, and pesticide residues).
Drug Analysis
GC is widely applied to identification of the active
components, possible impurities as well as the metabolites.
Aspects of GC Applications:

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Forensic Analysis
In forensic cases, very little sample is available, and the
concentration of the sample components may be very low.
GC is a useful due to its high sensitivity and separation efficiency.
Environmental Analysis
The environmental contaminants; e.g. dichlorodiphenyltrichloro-
ethane (DDT) and the polychlorinated biphenyls (PCBs) are present
in the environment at very low concentrations and are found among
many of other compounds.
GC, with its high sensitivity and high separating power, is mostly
used in the analysis of environmental samples.

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